ENVIRONMENTAL PROTECTION AGENCY

40 CFR Parts 110 and 300 

National Oil and Hazardous Substances Pollution Contingency Plan 

U.S. Environmental Protection Agency (EPA) Proposed rule.

Acute Toxicity Testing Protocol Including Procedures for Product/Oil Mixtures

The Agency is proposing provisions for conducting product-only acute toxicity tests with 

all agent categories (i.e., dispersants, surface washing agents, bioremediation agents, solidifiers, 

and herding agents). However, the Agency is proposing provisions for conducting acute toxicity 

tests with product-oil mixtures for dispersants only, and not for the other agent categories. EPA 

believes that non-dispersant products are not likely to be used in the same quantities or durations 

as dispersants and further, that some may be recovered. Thus, making a requirement to conduct 

toxicity tests of product/oil mixtures for agent categories other than dispersants may not be 

necessary. 

There may be concerns, however, for increased product toxicity when non-dispersant 

products interact with the oil. Thus, the Agency is requesting comment on the need, in addition 

to the proposed acute toxicity testing requirements for surface washing agents, bioremediation 

agents, solidifiers, and herding agents, to include requirements for product-reference oil mixtures 

for these categories. 

A protocol that includes procedures for the preparation of product/oil mixtures for 

toxicity testing of these other agent categories is provided below for review as an alternative to

the one included in the proposed rule. This revised protocol is consistent with the testing protocol 

proposed for dispersants. The Agency is requesting comments on the protocol itself, including 

the preparation procedures for the product-oil mixtures. The redlined text indicates changes to 

the proposed protocol that include the preparation of product/oil mixtures for toxicity testing. 

Specifically, the Agency is requesting comment on whether it should require testing of 

alternative product to oil ratios for non-dispersant products to reflect potentially higher product 

concentrations that may be recommended by the manufacturer.
4.0 Standard Acute Toxicity Testing of Surface Washing Agents, Bioremediation Agents, 

Herding Agents, and Solidifiers. 

4.1 Summary. This laboratory protocol includes testing for: 1) saltwater standard static acute 

toxicity tests for test products with the mysid shrimp, Americamysis bahia (48-hr duration) and 

the inland silverside, Menidia beryllina (96-hr duration); 2) test product/oil mixture static acute 

toxicity tests for Americamysis bahia and Menidia beryllina (48-hr and 96-hr duration, 

respectively); 3) freshwater standard static acute toxicity tests for test products with the daphnid, 

Ceriodaphnia dubia (48-hr duration) and the fathead minnow, Pimephales promelas (96-hr 

duration); and 4) test product/oil mixture static acute toxicity tests for Ceriodaphnia dubia and 

Pimephales promelas (48-hr and 96-hr duration, respectively) (see Table 8 of this Appendix).

TABLE 8 – TOXICITY TESTING REQUIREMENTS FOR SURFACE WASHING AGENTS, 

HERDING AGENTS, BIOREMEDIATION AGENTS AND SOLIDFIERS

Test Procedure

Application 

Environment

96-hr Static 

Acute: Menidia 

beryllina

48-hr Static 

Acute:

Americamysis 

bahia

96-hr Static 

Acute: 

Pimephales 

promelas

48-hr Static 

Acute: 

Ceriodaphnia 

dubia

Saltwater only yes yes no no

Freshwater only no no yes yes

Freshwater and 

saltwater use yes yes yes yes

4.2 Dilution Water. Use Section 7 of EPA’s Methods for Measuring the Acute Toxicity of 

Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA-821-R-02-012) [1]

for preparation of the appropriate dilution water for each species tested. Use of clean natural or 

synthetic seawater for tests conducted with saltwater species is acceptable. 

4.3 Preparation of Stock Solutions.

4.3.1 Liquid Surface Washing Agents and or Herding Agents. Prepare a 1000 μL/L stock 

solution prior to test initiation by adding 1.1 mL of test product to 1100 mL of dilution water in a 
glass vessel. Place on a magnetic stir plate then add and center a stir bar and adjust the stir plate 

to obtain a vortex of 25% of the total volume of the liquid. Mix the resulting stock solution for 

approximately five minutes at room temperature. Using a glass pipette, remove appropriate 

aliquots of stock solution from between the mixing vessel wall and edge of the vortex and place 

directly into the dilution water within an exposure vessel. Base the preparation of exposure 

solutions on the nominal concentration of the stock solution and follow procedures outlined in 

sections 4.6 and/or 4.7 of this Appendix, as appropriate.

4.3.2 Bioremediation Agents. For products consisting of two or more liquid and/or solid 

components, prepare the product following the manufacturers recommended procedure and 

ensure the test product mixture is completely blended. Prepare a 1000 μL/L stock solution prior 

to test initiation by adding 1.1 mL of the test product mixture to 1100 mL of dilution water in a 

glass vessel. Place on a magnetic stir plate then add and center a stir bar and adjust the stir plate 

to obtain a vortex of 25% of the total volume of the liquid. Mix the resulting stock solution for 

approximately five minutes at room temperature. Using a glass pipette, remove appropriate 

aliquots of stock solution from between the mixing vessel wall and edge of the vortex and place 

directly into the dilution water within an exposure vessel. Base the preparation of exposure 

solutions on the nominal concentration of the stock solution and follow procedures outlined in 

sections 4.6 and/or 4.7 of this Appendix, as appropriate.

4.3.3 Solid Phase Products. Assessment of the toxicity of solidifiers and other solid phase 

products are determined using the aqueous phase of water-accommodated fractions (WAFs) of 

the test product. Fit a glass aspirator bottle (approximately 23L) equipped with hose bib at the 

base with a length of silicon tubing containing a hose clamp. Fill the bottle with 19L of dilution 

water leaving a 20% headspace above the liquid, place on a magnetic stir plate then add and 
center a stir bar. Add the test product at 25 g/L and securely seal the bottle using a silicon stopper 

and wraps of parafilm. Adjust the stir plate to obtain a vortex of 25% of the total fluid volume, 

stir for 18 hours then settle for 6 hours. Maintain the temperature at 250 C during stirring and 

settling. Purge the hose at the base of the bottle of any material followed by removal of the 

WAF (aqueous phase) into a clean glass container without disturbing the product on the surface. 

The WAF should be remixed and used for the preparation of exposure solutions following 

procedures outlined in section 4.4 of this Appendix.

4.3.4 Test Product-Reference oil(s) Mixtures. Use IFO 120 and non-weathered ANS oils. 

To obtain these oils, see http://www.epa.gov/emergencies/content/ncp/index.htm. Assessment of 

test product-reference oil mixture toxicity is determined for each reference oil using the aqueous 

phase of a chemically enhanced-water accommodated fraction (CE-WAF). Repeat the following 

procedure for each reference oil tested. Fit a glass aspirator bottle (approximately 23L) equipped 

with hose bib at the base with a length of silicon tubing containing a hose clamp. Fill the bottle 

with 19L of seawater or freshwater, as appropriate, leaving a 20% headspace above the liquid, 

place on a magnetic stir plate then add and center a stir bar. Add the respective crude oil at 25 

g/L using a silicon tube attached to a glass funnel that reaches just below the water surface. 

Using this method reduces the production of air bubbles on the oil surface slick. Adjust the stir 

plate to obtain an oil vortex of 25% of the total volume of the seawater or freshwater, as 

appropriate, then add the product to be tested at a ratio of 1:10 product: oil (2.5 g/L). Securely 

seal the bottle to reduce the loss of volatiles using a silicon stopper and wraps of Parafilm and 

stir for 18 hours, then allow the solution to settle for 6 hours. Maintain the temperature at 250 C 

during stirring and settling. Purge the hose at the base of the bottle of any material followed by

removal of the CE-WAF (aqueous phase) into a clean glass container without disturbing the 
surface oil slick. The CE-WAF should be remixed and 1-2 L removed for chemical analysis of 

TPH following the procedures outlined in Section 4.5. The remaining volume will be used for 

the preparation of exposure solutions following procedures outlined in Section 4.4. To reduce 

time and cost, it is preferable to mix sufficient amounts of test product-reference oil mixture CEWAF to allow preparation of exposure solutions for conducting simultaneous acute tests with the 

two saltwater species and/or the two freshwater species, as appropriate.

4.4 Preparation of Exposure Concentrations.

4.4.1 Concentration Selection. Preliminary rangefinder tests may be necessary using a 

series of logarithmic concentrations (e.g. 0.1, 1, 10, 100 μl test product/L or mg TPH/L) to 

determine the appropriate exposure concentration range necessary to determine LC50 values and 

95% confidence intervals. For definitive tests, conduct a minimum of five test concentrations 

using a geometric ratio between 1.5 and 2.0 (e.g. 2, 4, 8, 16, and 32). Note that when testing only 

the test product, the highest test concentration should not exceed the test products selfdispersibility limit, with exception of solid phase products.

4.4.2 Exposure Concentrations. Exposure solutions are prepared by adding the 

appropriate amount of stock solution directly to dilution water in each test chamber. Mix each 

exposure solution using five rotations in one direction followed by five rotations in the opposite 

direction using a solid glass stir rod.

4.4.3 Reference Toxicants. Separate toxicity tests must be performed with a reference 

toxicant for each species tested. Conduct additional reference toxicity tests any time a change in 

the culture population or source of a test species occurs. Use reagent grade quality sodium 

dodecyl sulfate (SDS), also known as dodecyl sodium sulfate (DSS), and sodium lauryl sulfate 

(SLS) as the reference toxicant. Information on procedures for conducting reference toxicant 
tests with these species can be found in section 4 of EPA’s Methods for Measuring the Acute 

Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA-821-R02-012) [1].

4.5 Chemical Analysis of Stock Solutions. Add the 1L sample of CE-WAF (Section 4.3.d) 

solutions directly to amber glass bottles with Teflon®-lined cap. Collect a replicate sample in the 

event of accidental loss or if reanalysis of the stock solution becomes necessary. Adjust sample 

to a pH=2 using 50% hydrochloric acid, immediately refrigerate and analyze within 48 hours of 

collection. Analyze samples for C9-C32 total petroleum hydrocarbons (TPH) by gas 

chromatography-flame ionization detection (GC-FID) following EPA SW-846, Method 8015BDRO. Report TPH concentration of stock solutions as milligrams TPH/L and use in the 

calculation of exposure concentrations for all toxicity tests conducted with CE-WAF.

4.6 Saltwater Static Acute Tests with Menidia beryllina and Americamysis bahia

4.6.1 General. Use EPA’s Methods for Measuring the Acute Toxicity of Effluents and 

Receiving Waters to Freshwater and Marine Organisms (EPA-821-R-02-012) [1] for testing 

each species separately with the test product or a mixture of the test product and reference oil.

4.6.2 Test Solutions. Modify procedures in EPA-821-R-02-012 specifically dealing with 

the handling and toxicity testing of effluents or receiving water samples as follows: Prepare stock 

solutions following the appropriate sections (4.3.1, 4.3.2, or 4.3.3, and 4.3.4) and exposure 

concentrations following section 4.4 of this Appendix.

4.6.3 Number of Treatments, Replicates and Organisms. Conduct a minimum of three 

replicates of at least five exposure treatments plus a minimum of three replicate dilution water 

controls. Expose ten organisms per replicate treatment.
4.6.4 Exposure Period. Test duration is 48-hr for A. bahia and 96-hr for M. beryllina. 

Mortality must be recorded at each 24 hour period of each test.

4.6.5 Test Acceptability. For each test performed, survival of control animals must be > 

90% and test results must allow determination of statistically valid LC50 and 95% confidence 

interval values except in cases where the LC50 is >1000 μl/L or is determined to be greater than 

the limits of water solubility of dispersibility.

4.6.6 Static Acute Test Summary. A summary of required test conditions is provided in 

Table 9.

4.7 Freshwater Static Acute Tests with Pimephales promelas and Ceriodaphnia dubia

4.7.1 General. Use EPA’s Methods for Measuring the Acute Toxicity of Effluents and 

Receiving Waters to Freshwater and Marine Organisms (EPA-821-R-02-012) [1] for testing 

each species separately with the test product or a mixture of the test product and reference oil.

4.7.2 Test Solutions. Modify procedures in EPA-821-R-02-012 specifically dealing with 

the handling and toxicity testing of effluents or receiving water samples as follows: Prepare stock 

solutions following the appropriate sections (4.3.1, 4.3.2, or 4.3.3, and 4.3.4) and exposure 

concentrations following section 4.4 of this Appendix.

4.7.3 Number of Treatments, Replicates and Organisms. P. promelas: Conduct a 

minimum of three replicates of at least five exposure treatments plus a minimum of three 

replicate dilution water controls. Expose ten organisms per replicate treatment. C. dubia: 

Conduct a minimum of four replicates of at least five exposure treatments plus a minimum of 

four replicate dilution water controls. Expose five organisms per replicate treatment.

4.7.4 Exposure Period. Test duration is 48-hr for C. dubia and 96-hr for P. promelas. 

Mortality must be recorded at each 24 hour period of each test.
4.7.5 Test Acceptability. For each test performed, survival of control animals must be > 

90% and test results must allow determination of statistically valid LC50 and 95% confidence 

interval values except in cases where the LC50 is >1000 μl/L or is determined to be greater than 

the limits of water solubility of dispersibility.

4.7.6 Static Acute Test Summary. A summary of required test conditions is provided in 

Table 9.

4.8. Laboratory Report. The laboratory must include, for each toxicity test report, all applicable 

information, data and analyses as follow:

4.8.1 Test Objective: protocol title and source, endpoint(s);

4.8.2 Product Information: product name, manufacturer contact information, lot number, 

production date, date received / chain of custody;

4.8.3 Contract Facility: contact information;

4.8.4 Dilution Water: source, pretreatment, physical and chemical characteristics (pH, 

salinity);

4.8.5 Test Conditions: date and time of test (start and end), test chambers type and 

volume, volume of solution per chamber, number of organisms per chamber, number of replicate 

chambers per treatment, feeding frequency, amount and type of food, test concentrations, test 

temperature (mean and range), test salinity (mean and range);

4.8.6 Test Organisms: common and scientific name, source contact information, age and 

date purchased, acclimation conditions (e.g., temperature, salinity, both mean and range), age at 

test start;

4.8.7 Reference toxicant: date received, lot number, date of most recent test, results and 

current Cumulative Sum Chart, dilution water used, physical and chemical methods used;
4.8.8 Quality Assurance: verification of laboratory accreditation, including subcontractor 

facilities;

4.8.9 Test Results: raw data in tabular and graphical form, daily records of affected 

organisms in each concentration replicate and controls, table of required endpoints (i.e., LC50, 

95% CI, IC50, LOEC and NOEC), statistical methods used to calculate endpoints, summary 

tables of test conditions and QA data;

4.8.10 Analytical Results: method summary including Limit of Detection (LOD)/Limit of 

Quantitation (LOQ), deviations and reasons if any, sample summary, results including 

chromatograms and data qualifiers, QA summary including calibration curves, method blank and 

surrogate recovery, analytical results summary; and 

4.8.11 Conclusions: Relationship between test endpoints and threshold limit.
TABLE 9 – SUMMARY OF TEST CONDITIONS

Saltwater 

Acute 

M. beryllina

Saltwater 

Acute 

A. bahia

Freshwater 

Acute

P. promelas

Freshwater 

Acute

C. dubia

Test type Static nonrenewal

Static nonrenewalStatic nonrenewalStatic nonrenewal

Test duration 96 hours 48 hours 96 hours 48 hours

Salinity 20 ± 2‰ 20 ± 2‰ NA NA

Temperature

25 ± 10C (recommended). Test temperatures must not deviate from 

250C (maximum minus minimum temperature) by more than 30C 

during the test (required).

Light quality Ambient laboratory illumination

Light intensity 10-20 μE/m2

/s

Photoperiod 16 h light, 8 h darkness, with phase in/out period recommended

Test chamber size1 250 mL 250 mL 250 mL 30 mL

Test solution volume1 200 mL 200 mL 200 mL 15 mL

Age of test organism2 9-14 days 1-5 days 1-14 days <24 hours

No. organisms per test 

chamber 10 10 10 5

No. of replicate chambers 

per concentration 

(minimum)

3 3 3 4

Feeding regime Refer to specific feeding procedures provided in each test method

Aeration None, unless DO falls below 4.0 mg/L, then aerate all chambers. 

Rate:<100 bubbles/minute

Test concentrations 5 exposure concentrations and a control (minimum required)

Test acceptability 

(required) ≥90% survival in controls

1

Recommended minimum value. 2Less than or equal to 24-hr range in age.

4.9 References for Section 4

(1) U.S. EPA. 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving 

Waters to Freshwater and Marine Organisms. Fifth Edition. U.S. Environmental Protection 

Agency, Washington, DC (EPA 821-R-02-012).